© copyright 2009 by the american association for clinical chemistry enrichment and detection of...

© Copyright 2009 by the American Association for Clinical Chemistry

Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR

L. Zhou, R.A. Palais, G.D. Smith, D. Anderson, L.R. Rowe, and C.T. Wittwer

May 2010

http://www.clinchem.org/cgi/reprint/56/5/814


Journal ClubJournal Club


IntroductionIntroduction

In early and post treatment stages of cancer

Mutation allele fraction may be low (<10%)

Mutations may be undetectable by sequencing

Current enrichment methods have limitations

Scorpion primers (complex)

TaqMan (fluorescent labels)

PNA and LNA (specially modified oligos)

ARMS (false positives)


Introduction (cont)Introduction (cont)

Snapback primers

Closed-tube genotyping method

Use saturating dyes instead of covalent fluorescent labels

No special modifications

Only 2 primers are required One primer has a 5’-tail that hybridizes to its extension product


QuestionsQuestions

What is a snapback primer?

What structure does a snapback primer form after PCR?

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 1. PCR and melting analysis of snapback primers. Snapback primers are standard oligonucleotides that include as a probe element a 5’-tail that is complementary to the extension product of the primer. Both full-length amplicons and intramolecular hairpins are formed after PCR. Melting of the probe element of the snapback hairpin provides targeted genotyping, whereas high-resolution analysis of amplicon melting optionally detects variants anywhere within the PCR product.


Materials and MethodsMaterials and Methods

Mechanism of enrichment

Mismatch the rare allele to the probe element

Use polymerase without 5’-exonuclease activity

Completely matched wild type allele blocks extension

Mismatched rare allele amplifies more efficiently


Materials and Methods (cont)Materials and Methods (cont)

Snapback enrichment factors

Probe element length and Tm

Extension temperature

Extension time

Mg++ concentration


Materials and Methods (cont)Materials and Methods (cont)

Control human cell lines

BRAF p.600E homozygote

EGFR p.delE746-A750 homozygote

Clinical Samples

47 patients with thyroid tumors

8 patients with lung tumors


QuestionsQuestions

How do snapback primers enrich minority alleles?

What factors influence enrichment?


Figure 2. Mechanism of enrichment using snapback primers. When the PCR conditions are carefully chosen, the polymerase unfolds and extends the destabilized mutant hairpins, but wild type extension is blocked, resulting in enrichment of the mutant allele.


ResultsResults

Factors that increase minority allele enrichment with snapback primers

Longer snapback probe elements with higher Tms

Lower extension temperature

Shorter extension time

Lower Mg++ concentration


Figure 3. The effect of snapback probe element length and Tm on allele enrichment. In (A) the 9 bp probe element (Tm=64°C), shows balanced peaks without apparent allele enrichment. In (B), the 13 bp probe element (Tm=68°C) shows some preference for the mismatched allele. In (C), the 17 bp probe element (Tm=74°C) definitely enriches the mismatched allele.

A

B

C


Figure 4. The effect of extension temperature on allele enrichment with snapback primers. As the extension temperature is decreased from 76ºC (left panel) to 70ºC (right panel), the perfectly matched wild type allele in the heterozygote appears to disappear.

A/GGA

95°C 0s63°C 0s76°C 5s

95°C 0s63°C 0s70°C 5s


Figure 5. The effect of extension time on snapback primer allele enrichment. The extension times were varied between 0 and 20 s at a free Mg++ concentration of 1.2 mM. The shorter the extension time, the higher the apparent mutation fraction. The initial minor allele percentages were either 1% (circles) or 0.1% (triangles).


Figure 6. The effect of Mg++ concentration on snapback primer allele enrichment. The free Mg++ concentration was varied between 0.8 mM and 2.2 mM, with the extension temperature at 70°C for 0 s. The lower the Mg++ concentration, the higher the apparent mutation fraction. The initial minor allele percentages

were either 1% (circles) or 0.1% (triangles).


ResultsResults

Sensitivity of rare allele detection

0.1% for BRAF p.600E

0.02% for EGFR p.delE746-A750

Snapback primers and HybProbes correlate

R2 = 0.93

More mutations identified with snapback primers


QuestionQuestion

Do the results support the proposed mechanism of snapback primer enrichment?


Figure 7. Analysis of thyroid nodules for BRAF p.V600E with both dual hybridization probes and snapback primers after enrichment. In panel A (left), standard curves for dual hybridization probes (HybProbes®, triangles) and snapback primers (circles), correlate the apparent mutation fraction to the actual mutation fraction. In panel B (right), the standard curves of each method are used to compare the actual mutation fractions of 91 thyroid samples using both HybProbe and snapback primer analysis.


DiscussionDiscussion

Snapback primer genotyping can be modified to enrich minor alleles in tumor samples

High probe element Tm relative to extension temperature

Short extension times

Low Mg++ concentration


Discussion (cont)Discussion (cont)

Rapid cycle PCR enables snapback primer enrichment

PCR efficiency of mutation mismatch > wild type match

70 cycles, although excessive by typical standards, is completed within 25 min

© copyright 2009 by the american association for clinical chemistry enrichment and detection of...

Documents